A strain of Trichoderma wumengense GUWM22 and its application

Abstract

The present invention discloses a strain of Trichoderma wumengense GUWM22 and its application, which belong to the field of microorganisms. The deposit number of the Trichoderma wumengense GUWM22 is CCTCC NO: M20242639, the deposit date is November 26, 2024, the deposit unit is the China Center for Type Culture Collection, and the deposit address is Wuhan University, Wuhan, China. It has been verified that the GUWM22 bacteria have a strong inhibitory effect on 6 pathogens (Phytophthora nicotianae, Phytophthora capsici, Botrytis cinerea, Alternaria alternata, Phomopsis simiana and Echinops planifolia), and the inhibition rate is more than 78%. In addition, GUWM22 also has a good prevention and control effect on post-harvest anthracnose of pepper (strip pepper), with a prevention effect of 82.11%. It can be seen that Trichoderma wumengense GUWM22 is a new species of biocontrol Trichoderma with good potential. The present invention provides a new strain resource and an environmentally friendly and efficient biocontrol approach for the effective prevention and control of pepper anthracnose.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a strain of Trichoderma wumengense GUWM22 and applications thereof. Background Art

[0002] Plant diseases cause significant economic losses annually. Currently, chemical control remains the primary method for plant disease prevention. However, the continued and extensive use of chemical pesticides has led to serious "3R" problems: increased resistance among pathogens, pesticide residues in the soil and environment, and the resurgence of pathogenic microorganisms. This not only threatens human health but also poses serious risks to the environment and non-target organisms.

[0003] Biological control is a key technology for green crop pest control. Biocontrol involves screening and utilizing beneficial microorganisms to create biological agents for use in preventing and controlling plant diseases and insect pests, thereby achieving disease control. It represents a new approach and method for green pest control. Microbial products are safe, sustainable, broad-spectrum, and environmentally friendly. Microbial agents work through mechanisms including antibiotics, competition, hyperparasitism, and the induction of systemic resistance in plants. They can also promote plant growth and increase agricultural yields. Fertilizers prepared using microorganisms can reduce the use of chemical fertilizers and pesticides by 30% to 60%, increase crop yields by 5% to 40%, and enhance plant resistance to pests and diseases, fundamentally reducing the use of chemical pesticides.

[0004] Currently, there are reports of a variety of microbial agents being used in production both domestically and internationally, including Bacillus subtilis, Trichoderma harzianum, Pseudomonas lilacinus, and Bacillus megaterium. The global market for biopesticides is also on the rise, and my country also has a substantial market for microbial agents. To develop multifunctional microbial agents, screening beneficial microorganisms from diverse environments provides a crucial source of microbial resources for product development. The Wumeng Grassland, one of the highest and largest karst plateau grasslands in Southwest China, possesses a unique ecological environment. Biocontrol bacteria in this unique environment must possess specialized adaptive mechanisms for extreme conditions, potentially possessing greater adaptability than those isolated from non-adverse environments, such as farmland systems. The unique ecological environment of the karst plateau provides a platform for the research, development, and application of biocontrol bacteria adapted to plateau environments. Summary of the Invention

[0005] The present invention provides a strain of Trichoderma wumengense GUWM22 and its use to address the problems of the prior art. GUWM2 exhibits an inhibition rate of up to 89.52% against pepper anthracnose pathogens and an 82.11% efficacy against postharvest anthracnose in peppers. This provides a new bacterial strain resource and an environmentally friendly, efficient biocontrol approach for the effective prevention and treatment of pepper anthracnose.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a strain of Trichoderma wumeng GUWM22, the preservation number of the Trichoderma wumeng GUWM22 is CCTCC NO: M20242639, the preservation date is November 26, 2024, the preservation unit is the China Center for Type Culture Collection, and the preservation address is Wuhan University, Wuhan, China.

[0008] The present invention also provides the use of the Trichoderma wumengmeng GUWM22 or its spores in inhibiting pathogens.

[0009] The present invention also provides the use of the Trichoderma wumengmeng GUWM22 or its spores in preparing a bacterial agent for inhibiting pathogens.

[0010] Optionally, the pathogens include Phytophthora nicotianae, Phytophthora capsici, Botryosphaeria dothidea, Alternaria alternata, Phomopsis sp and Colletotrichum truncatum.

[0011] The present invention also provides the use of the Trichoderma wumengmeng GUWM22 or its spores in preventing and / or treating pepper anthracnose.

[0012] The present invention also provides the use of the Trichoderma wumengmeng GUWM22 or its spores in preparing a fungal agent for preventing and / or treating pepper anthracnose.

[0013] The present invention also provides a fungal agent for preventing and / or treating pepper anthracnose, comprising the Trichoderma wumengense GUWM22 or its spores.

[0014] The present invention also provides a method for preventing and controlling pepper anthracnose, comprising the step of inoculating pepper fruits with the spore suspension of Trichoderma wumengense GUWM22.

[0015] Optionally, the pathogen of pepper anthracnose includes Colletotrichum truncatum.

[0016] The present invention discloses the following technical effects:

[0017] A new strain of Trichoderma was isolated from the Wumeng grasslands in Guizhou Province. Based on morphological and multi-gene phylogenetic identification, this strain is a new species of Trichoderma in the clade Harzianum and is named Trichoderma wumeng GUWM22. This strain was deposited with the China Center for Type Culture Collection on November 26, 2024, with the accession number CCTCCNO: 20242639. A plate standoff experiment using GUWM22 and six highly pathogenic plant pathogens (Phytophthoranicotianae, Phytophthora capsici, Botryosphaeria dothidea, Alternaria alternata, Phomopsis sp., and Colletotrichum truncatum) revealed that the bacterium had a strong inhibitory effect against all six pathogens, with inhibition rates exceeding 78%. The highest inhibition rate was 89.52% against pepper anthracnose. Furthermore, GUWM22 demonstrated excellent control efficacy against postharvest anthracnose of peppers (bell peppers), achieving an efficacy of 82.11%. This indicates that GUWM22 is a promising new biocontrol Trichoderma species. The present invention provides a new bacterial strain resource and an environmentally friendly, efficient biocontrol approach for the effective control of pepper anthracnose. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 is the phylogenetic tree of strain GUWM22;

[0020] Figure 2 Figure 5 shows the morphological characteristics of strain GUWM22; AB: morphology of the front and back of the colony cultured on PDA medium at 25°C for 7 days; C: conidial pustules on PDA; D: chlamydospores; E: conidia; FH: conidiophores and phialides;

[0021] Figure 3 The colony morphology of six pathogens cultured against strain GUWM22;

[0022] Figure 4 The inhibition rate of six pathogens in confrontation culture with strain GUWM22;

[0023] Figure 5 Symptom phenotypes of pepper fruits on the 7th day after inoculation with C. truncatum; A and C: control groups; B and D: treatment groups;

[0024] Figure 6 The diameter of the lesion on pepper fruit 7 days after inoculation with C. truncatum. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] Example 1 Isolation, screening and identification of Trichoderma

[0031] 1. Separation and screening

[0032] 1.1 Test samples

[0033] Root soil samples of Quercus alpina and Rhododendron were collected from Wumeng Prairie (104°37′E, 26°11′N), Liupanshui City, Guizhou Province in August 2023.

[0034] 1.2 Main Reagents

[0035] Potato dextrose agar (PDA): 200 g potatoes (peeled), 20 g glucose, 15-20 g agar powder, and 1000 mL distilled water.

[0036] 1.3 Isolation, screening, purification and preservation of Trichoderma

[0037] Using the dilution plate method, 1 g of each soil sample was placed in a 9 mL sterile water test tube and diluted to 10 -1 , 10 -2 , 10 -3 Concentration, take 100 μL of each dilution and spread on PDA plates. Repeat three times for each concentration. Incubate in the dark at 25°C in a constant temperature incubator. After colonies grow, pick mycelial blocks from the edge of the colony and transfer them to new PDA medium for purification. Select colonies with morphology similar to Trichoderma for storage. Label the purified strains with numbers and store the cakes in cryovials containing a final concentration of 15% glycerol at -80°C. Alternatively, the strains can be cultured on PDA slants and stored in a room temperature chamber.

[0038] Results: A strain with a colony morphology consistent with that of Trichoderma spp. was obtained.

[0039] 2. Construction of Trichoderma molecular phylogenetic tree

[0040] 2.1 Trichoderma DNA extraction

[0041] The strain was cultured on PDA medium for 7 days (25°C), and then DNA was extracted using the Ezup column fungal DNA extraction kit (Shanghai Sangon, China) according to the instructions.

[0042] 2.2 PCR amplification

[0043] The ITS universal primer pair (ITS4 and ITS5), RPB2 primer pair (fRPB2-5f and fRPB2-7cr), and TEF1 primer pair (EF1-728F and TEF1LLErev) synthesized by Beijing Qingke Biotechnology (Chongqing) Technology Co., Ltd. were used to perform PCR on the strain gene sequence. The specific primer information is shown in Table 1.

[0044] Table 1 Primer information

[0045]

[0046] The extracted DNA was used as a template for PCR amplification. The PCR reaction system consisted of 25 μL of the following: 12.5 μL Taq PCR Master Mix, 9.5 μL ddH₂O, 1 μL forward and reverse primers, and 1 μL DNA template. A 1% agarose gel was prepared, and 2 μL of the PCR product was placed in the wells of the 1% agarose gel for electrophoresis. After approximately 30 minutes, when the bands converged to the center of the gel, the gel was removed and observed on a UV gel imager. The brightest bands in the gel were sent to Beijing Qingke Biotechnology (Chongqing) Co., Ltd. for sequencing.

[0047] SEQ ID NO.1 (GUWM22 ITS sequence):

[0048] ;

[0049] SEQ ID NO. 2 (GUWM22 TEF1 sequence):

[0050]

[0051] SEQ ID NO.3 (GUWM22 RPB2 sequence):

[0052]

[0053] 2.3 System evolution analysis

[0054] The nucleotide sequences sequenced by the company were aligned using BLAST in the NCBI database, and the corresponding nucleotide sequences of Trichoderma strains with high homology were downloaded from the GenBank database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) as reference sequences. The alignment was performed using the MAFFT v.7 online server (http: / / mafft.cbrc.jp / alignment / server / index) and manually adjusted using BioEdit v_7.2.6.1 (http: / / www.mbio.ncsu.edu / BioEdit / page2.html). The aligned gene sequences were spliced ​​according to the order of RPB2 and TEF1. A phylogenetic tree was constructed using IQ-TREE v.1.4.3, and maximum likelihood (ML) and Bayesian inference (BI) were used for analysis. The final phylogenetic tree was visualized using FigTree.

[0055] The phylogenetic analysis included 23 strains of Trichoderma (strain gene accession numbers are shown in Table 2), as well as two outgroup species, T. pingquanense. Because the ML and BI phylogenetic trees showed similar topological structures, only the ML tree is shown, see Figure 1 , revealing the taxonomy of 1 new species in the Harzianum clade.

[0056] Table 2 Strain gene accession numbers

[0057]

[0058] 3. Observation of morphological characteristics of Trichoderma

[0059] Strain GUWM22 was cultured at 25°C in the dark for 3 days. Mycelial masses at the edge of the colony were picked and transferred to the center of a new PDA plate and cultured at 25°C in the dark for three times. The growth of the colony on the PDA medium (including mycelial density, colony color, and conidia production) was recorded daily. After culturing on PDA medium for 2 days, a sterilized coverslip was inserted at a 45° angle at the edge of the colony. Once the mycelium grew onto the coverslip, the morphology of conidiophores, phialides, conidia, and chlamydospores was observed using a Zeiss microscope (Carl Zeiss Microscopy GmbH, Jena, Germany).

[0060] Observation results are shown in Figure 2Strain GUWM22 produced colonies on PDA medium without pigment or noticeable odor, but with abundant, flocculent aerial hyphae. After three days of culture, dark green conidia formed on the cake, primarily near the cake, with fewer clusters near the edges. After seven days, the entire dish was densely covered with conidia, with spores near the cake appearing dark green. On PDA medium, the colony grew rapidly, filling a 90 mm diameter dish in just two to three days at 25°C.

[0061] Conidiophores have a distinct main axis with paired or unilateral lateral branches sparsely arranged. Branches produce fewer phialides, which are not completely paired but bottle-shaped, solitary or in whorls of 2-3, measuring (5.3-13.7) × (1.6-3.5) μm, with an aspect ratio of 2-6.1 μm and a base width of 1.4-2.7 μm (n=50). Conidia are green, elliptical, measuring (2.7-4.2) × (2.3-3.9) μm, with an aspect ratio of 1-1.49 μm (n=50). Thick-walled, nearly spherical spores are terminal or intercalary, measuring (5.0-9.2) × (4.8-8.7) μm, with an aspect ratio of 1-1.49 μm (n=50).

[0062] Based on the results of multi-gene phylogenetic analysis of the isolated strain and its morphological characteristics, strain GUWM22 was identified as a new species of Trichoderma of the Harzianum branch, and was taxonomically named Trichoderma wumeng. It was deposited in the China Center for Type Culture Collection on November 26, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCCNO: M20242639.

[0063] Example 2 Determination of antibacterial ability against 6 pathogens

[0064] The antagonistic effect of strain GUWM22 against pathogens (see Table 2, provided by the Plant Pathology Laboratory, College of Agriculture, Guizhou University) was determined using the plate standoff method. Activated strain GUWM22 and pathogen cakes (5 mm in diameter) were inoculated on opposite sides of a PDA plate, 4 cm apart. A PDA plate inoculated with the pathogen alone served as a control. The plates were incubated at 25°C, and the colony diameters were recorded after 7 days. The antibacterial effect of strain GUWM22 was evaluated by calculating its inhibition rate against the pathogens. The inhibition rate was calculated as follows: Inhibition rate = [(CT) / C] × 100%. C and T represent the average growth radius of the pathogen in the control and treatment groups, respectively.

[0065] Table 2 Pathogens

[0066]

[0067]

[0068] Through the confrontation plate test, it was found that after 7 days of culture ( Figure 3 ), strain GUWM22 had a strong antibacterial effect on 6 pathogens, with an inhibition rate of more than 78% ( Figure 4 ), with inhibition rates of 78.89% against P. nicotianae, 79.95% against P. capsici, 85.31% against A. alternata, 85.99% against B. dothidea, 86.48% against Phomopsis sp., and 89.52% against C. truncatum. After seven days of co-culture, significant pigment changes were observed at the junctions between the hyphae of the six pathogens and strain GUWM22. In particular, C. truncatum exhibited significant sporulation at the junctions between T. wumeng and C. truncatum.

[0069] Example 3 Protective effect of spore suspension on pepper fruit

[0070] Through antagonistic experiments, the strain GUWM22, which has the best antibacterial effect, was selected as the research object to evaluate the protective effect of the strain against anthracnose of peppers (bell peppers). Sterile water was used as the control group, and the spore suspension of the strain GUWM22 was used as the treatment group. The spore suspension of Trichoderma truncatum (1×10 8 Spray the punctured area with 100 spores / mL (0.05 spores / mL) using sterile water as a control. The peppers were then placed in a 25°C incubator. Two days after inoculation, C. truncatum spores were inoculated into the punctured area. Seven days after inoculation, the fruit was observed for disease progression and the lesion diameter was measured using the cross-hatch method. Five peppers were sampled for each treatment, and the experiment was replicated three times. Disease prevention efficacy (%) = [(lesion diameter of control group - lesion diameter of treatment group) / lesion diameter of control group] × 100.

[0071] In this experiment, the disease control effect of the spore suspension of strain GUWM22 was determined to evaluate the disease control potential of strain GUWM22 on post-harvest peppers. Figure 5 As shown in the figure, 7 days after inoculation with C. truncatum cake, peppers in the control group all became diseased. C. truncatum produced yellow-brown lesions on the wounds of the fruits. There were a lot of white hyphae on the surface of the lesions. The diameter of the lesions ( Figure 5 Middle A, Figure 5 C); Compared with the control group, the peppers in the treatment group had basically no obvious lesion symptoms on the wounds of the fruits, and basically no mycelium on the lesions ( Figure 5 Middle B, Figure 5 Middle D).

[0072] The diameter of the lesions was calculated. On the 7th day after inoculation with C. truncatum, the average diameters of the lesions on peppers in the control group and the treatment group were 16.38 mm and 2.93 mm, respectively. There was a significant difference between the control group and the treatment group (P < 0.01). Figure 6 ).

[0073] The test results show that the spore suspension of Trichoderma wumengense GUWM22 obtained in the present invention has a good disease prevention effect on pepper fruits, with a prevention efficiency of 82.11%.

[0074] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A strain of Trichoderma wumengense ( Trichoderma wumeng ) GUWM22, characterized in that The preservation number of the Wumeng Trichoderma GUWM22 is CCTCC NO: M20242639, the preservation date is November 26, 2024, the preservation unit is the China Center for Type Culture Collection, and the preservation address is Wuhan University, Wuhan, China.

2. The use of Trichoderma wumengmeng GUWM22 or its spores in inhibiting pathogens as claimed in claim 1, characterized in that, The pathogen is Phytophthora nicotianae ( Phytophthora nicotianae )、Phytophthora capsici( Phytophthora capsici ), Botrytis cinerea ( Botryosphaeria dothidea ), Alternaria alternata ( Alternaria alternata )、Phomopsis fasciata( Phomopsis sp. ) and planocephalides ( Colletotrichum truncatum ).

3. The use of Trichoderma wumengmeng GUWM22 or its spores in the preparation of a microbial agent for inhibiting pathogens as claimed in claim 1, characterized in that: The pathogen is Phytophthora nicotianae ( Phytophthora nicotianae )、Phytophthora capsici( Phytophthora capsici ), Botrytis cinerea ( Botryosphaeria dothidea ), Alternaria alternata ( Alternaria alternata )、Phomopsis fasciata( Phomopsis sp. ) and planocephalides ( Colletotrichum truncatum ).

4. Use of the Trichoderma wumengmeng GUWM22 or its spores as claimed in claim 1 in preventing and / or treating pepper anthracnose.

5. Use of the Wumeng Trichoderma GUWM22 or its spores as claimed in claim 1 in preparing a fungal agent for preventing and / or treating pepper anthracnose.

6. A fungal agent for preventing and / or treating pepper anthracnose, characterized in that: It comprises the Trichoderma wumengmeng GUWM22 or its spores according to claim 1.

7. A method for preventing and treating pepper anthracnose, characterized in that: The method comprises the step of inoculating pepper fruits with the spore suspension of Trichoderma wumengense GUWM22 according to claim 1.

8. The method according to claim 7, wherein The pathogens of pepper anthracnose include flat-headed thorny fungi ( Colletotrichum truncatum ).

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